Title: Distinct Glycoprotein Ib/V/IX and Integrin αIIbβ3-dependent Calcium Signals Cooperatively Regulate Platelet Adhesion under Flow
Abstract: We have investigated the calcium signaling relationship between the two major platelet adhesion receptors, glycoprotein Ib/V/IX (GPIb/V/IX) and integrin αIIbβ3, involved in regulating platelet adhesion on von Willebrand factor (vWf) under flow. Our studies demonstrate that GPIb engagement of immobilized vWf elicits a transient calcium spike that may function to promote reversible arrest of translocating platelets. Subsequent integrin αIIbβ3 engagement of vWf promotes sustained calcium oscillations that are essential for the maintenance of irreversible adhesion. GPIb-induced calcium spikes appear distinct from those initiated by integrin αIIbβ3, in that the former are exclusively mediated through release of intracellular calcium stores via a signaling mechanism independent of PI 3-kinase. In contrast, integrin αIIbβ3-dependent calcium flux involves a PI 3-kinase-dependent signaling mechanism linked to intracellular calcium mobilization and subsequent transmembrane calcium influx. Studies employing the caged calcium chelator (o-nitrophenyl-EGTA) demonstrate that transient calcium spikes initiate a transient phase of platelet arrest that is converted to irreversible adhesion with the development of sustained oscillatory calcium flux. These studies demonstrate the existence of a dual step calcium signaling mechanism utilized by GPIb and integrin αIIbβ3 that serves to regulate the dynamics of platelet adhesion under flow. We have investigated the calcium signaling relationship between the two major platelet adhesion receptors, glycoprotein Ib/V/IX (GPIb/V/IX) and integrin αIIbβ3, involved in regulating platelet adhesion on von Willebrand factor (vWf) under flow. Our studies demonstrate that GPIb engagement of immobilized vWf elicits a transient calcium spike that may function to promote reversible arrest of translocating platelets. Subsequent integrin αIIbβ3 engagement of vWf promotes sustained calcium oscillations that are essential for the maintenance of irreversible adhesion. GPIb-induced calcium spikes appear distinct from those initiated by integrin αIIbβ3, in that the former are exclusively mediated through release of intracellular calcium stores via a signaling mechanism independent of PI 3-kinase. In contrast, integrin αIIbβ3-dependent calcium flux involves a PI 3-kinase-dependent signaling mechanism linked to intracellular calcium mobilization and subsequent transmembrane calcium influx. Studies employing the caged calcium chelator (o-nitrophenyl-EGTA) demonstrate that transient calcium spikes initiate a transient phase of platelet arrest that is converted to irreversible adhesion with the development of sustained oscillatory calcium flux. These studies demonstrate the existence of a dual step calcium signaling mechanism utilized by GPIb and integrin αIIbβ3 that serves to regulate the dynamics of platelet adhesion under flow. glycoprotein Ib/V/IX von Willebrand factor 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester platelet wash buffer thapsigargin phosphatidylinositol 3-kinase o-nitrophenyl-EGTA dimethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid The platelet is a specialized adhesive cell that plays a central role in the normal blood clotting process through its ability to rapidly adhere to subendothelial matrix proteins and to other activated platelets at sites of vascular injury. Platelet adhesion is a multistep process requiring an initial cell-tethering step, involving interaction between the glycoprotein Ib/V/IX (GPIb/V/IX)1 receptor complex with subendothelial von Willebrand factor (vWf) and a firm adhesion step mediated by one or more platelet integrins (1Savage B. Saldivar E. Ruggeri Z.M Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 2Savage B. Almus-Jacobs F. Ruggeri Z. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). Integrins themselves are generally inefficient at initiating platelet adhesion under conditions of rapid blood flow, due to their slow intrinsic binding kinetics. In contrast, the rapid formation and dissolution of bonds between the vWf A1 domain and GP Ibα supports efficient platelet tethering and translocation (rolling) under conditions of high shear (3Ruggeri Z.M Tromb. Haemost. 1997; 78: 611-616Crossref PubMed Scopus (324) Google Scholar). Understanding the mechanisms regulating platelet translocation is potentially important, since recent studies have demonstrated that the majority of platelets adhering to the injured vessel wall and to the surface of thrombi in vivo undergo a variable period of surface translocation prior to forming stationary adhesion contacts (4Denis C. Methia N. Frenette P.S. Rayburn H. Ullman-Cullere M. Hynes R.O. Wagner D.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9524-9529Crossref PubMed Scopus (434) Google Scholar, 5Kulkarni S. Dopheide S.M. Yap C.L. Ravant C. Freund M. Mangin P. Heel K.A. Street A. Harper I.S. Lanza F. Jackson S.P J. Clin. Inv. 2000; 105: 783-791Crossref PubMed Scopus (299) Google Scholar). There is a considerable body of evidence demonstrating that in addition to mediating platelet tethering and translocation, GPIb transduces signals necessary for integrin αIIbβ3activation. The mechanism by which GPIb transduces signals remains controversial and appears to be significantly influenced by the experimental conditions used to study this process. For example, based on studies of shear-induced platelet aggregation using a cone-and-plate viscometer, GPIb has been proposed to initiate platelet activation by inducing transmembrane calcium influx, leading to integrin αIIbβ3 activation through an indirect mechanism dependent on released ADP (6Moake J.L. Turner N.A. Staphopoulos N.A. Hellums J.D Blood. 1988; 71: 1366-1374Crossref PubMed Google Scholar). In contrast, recent studies examining platelet adhesion to an immobilized vWf matrix have demonstrated the existence of a distinct GPIb signaling mechanism linked to intracellular calcium mobilization (7Yuan Y. Ulsemer P. Cranmer S.L. Yap C.L. Nesbitt W.S. Harper I. Mistry N. Dopheide S.M. Hughan S.C. Williamson D. de la Salle C. Salem H.H. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 36241-36251Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). GPIb-dependent calcium mobilization is a shear-sensitive signaling process promoting integrin αIIbβ3activation directly, independent of released ADP. An important unresolved issue is the relative contribution of GPIb and integrin αIIbβ3 outside-in signaling to cytosolic calcium flux initiated by the platelet-vWf interaction. For example, a recent study by Kuwahara et al. has suggested that calcium flux during shear-dependent platelet adhesion on vWf is exclusively mediated through integrin αIIbβ3 (8Kuwahara M. Sugimoto M. Tsuji S. Miyata S. Yoshioka A. Blood. 1999; 94: 1149-1155Crossref PubMed Google Scholar). The involvement of integrins in the regulation of cytosolic calcium levels has been well established in a range of cell types, including platelets, leukocytes, endothelial cells, fibroblasts, and osteoclasts (9Sjaastad M.D. Lewis R.S. Nelson W.J. Mol. Biol. Cell. 1996; 7: 1025-1041Crossref PubMed Scopus (62) Google Scholar). In general, calcium signaling is utilized by β2 leukocyte integrins as well as integrins that engage ligands containing the tripeptide sequence Arg-Gly-Asp (RGD), including platelet αIIbβ3, αvβ5, and αvβ3 (9Sjaastad M.D. Lewis R.S. Nelson W.J. Mol. Biol. Cell. 1996; 7: 1025-1041Crossref PubMed Scopus (62) Google Scholar, 10Pelletier A.J. Bodary S.C. Levinson A.D. Mol. Biol. Cell. 1992; 3: 989-998Crossref PubMed Scopus (90) Google Scholar, 11Francesconi M. Casonato A. Pontara E. Dalla V.L. Girolami A. Deana R. Biochem. Biophys. Res. Commun. 1995; 214: 102-109Crossref PubMed Scopus (27) Google Scholar, 12Schwartz M.A. J. Cell Biol. 1993; 120: 1003-1012Crossref PubMed Scopus (219) Google Scholar, 13Schwartz M.A. Deninghoff K. J. Biol. Chem. 1994; 269: 11133-11137Abstract Full Text PDF PubMed Google Scholar, 14Sjaastad M.D. Angres B. Lewis R.S. Nelson W.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8214-8218Crossref PubMed Scopus (47) Google Scholar). However, the relationship between integrins and calcium signaling is complex and in most cell types incompletely understood. For example, in platelets there is evidence that αIIbβ3 plays an important role in regulating calcium homeostasis in the resting cell and promotes calcium influx following platelet activation (15Brass L.F. J. Biol. Chem. 1985; 260: 2231-2236Abstract Full Text PDF PubMed Google Scholar, 16Sage S.O. Sargeant P. Heemskerk J.W. Mahaut-Smith M.P Adv. Exp. Med. Biol. 1993; 344: 69-82Crossref PubMed Scopus (11) Google Scholar). Other studies have suggested that ligand binding to αIIbβ3 can either induce or inhibit calcium mobilization, depending on the nature of the primary platelet-activating stimulus (17Heemskerk J.W.M. Hoyland J. Mason W.T. Sage S.O. Biochem. J. 1992; 283: 379-383Crossref PubMed Scopus (48) Google Scholar, 18Chang H.H. Lin C.H. Lo S.J. Exp. Cell Res. 1999; 250: 387-400Crossref PubMed Scopus (33) Google Scholar, 19Rosado J.A. Meijer E.M.Y. Hamulyak K. Novakova I. Heemskerk J.W.M. Sage S.O. Blood. 2001; 97: 2648-2656Crossref PubMed Scopus (31) Google Scholar). In this study, we have examined the relative contribution of GPIb and integrin αIIbβ3 in promoting cytosolic calcium flux during shear-dependent platelet adhesion on immobilized vWf. We demonstrate the existence of a dual step calcium signaling mechanism initiated by vWf engagement of GPIb and integrin αIIbβ3 that serves to regulate platelet translocation dynamics and firm platelet adhesion under flow. 5,5′-Dimethyl-BAPTA, AM; Oregon Green 488 BAPTA-1, AM; Fura Red, AM; and NP-EGTA were from Molecular Probes, Inc. (Eugene, OR). Apyrase was purified from potatoes according to the method of Molnar and Lorand (20Molnar J. Lorand L.A. Biochem. Biophys. Acta. 1961; 93: 353-363Google Scholar) and was a generous gift from Dr. Francois Lanza. Human vWf was purified to homogeneity from plasma cryoprecipitate according to the methods of Montgomery and Zimmerman (21Montgomery R.R. Zimmerman T.S.J J. Clin. Invest. 1978; 61: 1498-1507Crossref PubMed Scopus (78) Google Scholar). LY294002 was from Calbiochem. Wortmannin was purchased from Sapphire Bioscience P/L (New South Wales, Australia). All other reagents were obtained from sources described previously (22Yuan Y. Dopheide S.M. Ivanidis C. Salem H.H. Jackson S.P J. Biol. Chem. 1997; 272: 21847-21854Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 23Cranmer S.L. Ulsemer P. Cooke B.M. Salem H.H. de la Salle C. Lanza F. Jackson S.P J. Biol. Chem. 1999; 274: 6097-6106Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Anticoagulated whole blood (15 mm trisodium citrate, pH 7.4) was collected from healthy volunteers who had not received any anti-platelet medication in the preceding 2 weeks. Platelet isolation was carried out according to Yuan et al.(22Yuan Y. Dopheide S.M. Ivanidis C. Salem H.H. Jackson S.P J. Biol. Chem. 1997; 272: 21847-21854Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), and washed platelets were finally resuspended in modified Tyrode's buffer (10 mm HEPES, 12 mmNaHCO3, pH 7.4, 137 mm NaCl, 2.7 mmKCl, 5 mm glucose) supplemented with 1 mmCaCl2/MgCl2 or 1 mmEGTA/MgCl2 where indicated. Autologous red blood cells were prepared according to Yap et al. (24Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Guiliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) prior to reconstitution with isolated platelets (50% (v/v) hematocrit) in the presence of 0.4 units/ml apyrase (ADPase activity) and 1 unit/ml hirudin. Platelet calcium flux was monitored as previously described by Yap et al. (24Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Guiliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) Isolated platelets (1.5 × 109/ml) suspended in platelet wash buffer (PWB) (4.3 mmK2HPO4, 4.3 mmNa2HPO4, 24.3 mmNaH2PO4, pH 6.5, 113 mm NaCl, 5.5 mm glucose, 0.5% (w/v) bovine serum albumin, 10 mm theophylline) were incubated for 30 min at 37 °C with Oregon Green BAPTA-1, AM (1 μm) and Fura Red, AM (1.25 μm). Unincorporated dye was removed by washing twice with PWB. Platelets were subsequently resuspended in Tyrode's buffer containing either 1 mm CaCl2/MgCl2or 1 mm EGTA/MgCl2. Real time platelet calcium flux was monitored via confocal microscopy as the ratio of Oregon Green BAPTA-1 fluorescence over Fura Red fluorescence (with emission wavelengths of 500–570 and 600–710 nm, respectively). Fluorescence ratios were converted to relative cytosolic calcium concentration (Δ[Ca2+]c) according to Equation 1,Δ[Ca2+]c=170×(R−Rmin)/(Rmax−R)×Fmax/FminEquation 1 where 170 is the Kd value of Oregon Green BAPTA-1 Ca2+ binding; R represents the measured fluorescence ratio; Rmax is the mean fluorescence ratio determined from 200–400 cells suspended in Tyrode's buffer supplemented with 5 mm A23187 + 10 mm CaCl2; Rmin is the mean fluorescence ratio determined from 200–400 cells preincubated with 70 μm DM-BAPTA, AM (incubated for 30 min at 37 °C) and resuspended in Tyrode's buffer supplemented with 2 mm EGTA; Fmax andFmin representing the mean fluorescence values (arbitrary units) of Oregon Green BAPTA-1 forRmax and Rmin, respectively. The calculated calcium flux values are designated Δ[Ca2+]c to indicate that all calcium concentration estimates are relative to a zero point, set by DM-BAPTA calcium chelation. Calcium flux recordings were analyzed off-line using Leica Physiology Software (Leica TCS SP; Leica, Heidleberg, Germany), in the case of single cell recordings. Alternatively, platelet Δ[Ca2+]c was analyzed at a population level using MCIDTM Image analysis software (Imaging Research Inc., Ontario, Canada). The first 3 min of platelet flow was captured as sequential 37.5-s series (0.586 frames per second capture rate) via confocal microscopy, and individual frames were analyzed for fluorescence pixel intensities at 5.86-s intervals following background subtraction of small pixel targets of less than 10-pixel diameters. Data analyzed in this way were pooled and presented as a time-averaged picture as either a scatter plot or relative frequency histogram. It should be noted that population analysis done in this way represents a frequency profile of cytosolic calcium events rather than single-cell calcium recordings. Flow assays were performed using glass microcapillary tubes (Microslides, Vitro Dynamics Inc., NJ) coated with Human vWf (100 μg/ml) according to a modified method of Cooke et al. (25Cooke B.M. Usami S. Perry I. Nash G.B. Microvasc. Res. 1993; 45: 33-55Crossref PubMed Scopus (175) Google Scholar). Oregon Green BAPTA-1/Fura Red loaded platelets were reconstituted with washed red blood cells (50% hematocrit) and Tyrode's supplemented with either 1 mmCa2+/Mg2+ or EGTA/Mg2+ and perfused through vWf microcapillary tubes blocked with 10% heat-inactivated human serum at a shear rate of 1,800·s−1 or 10,000·s−1. Platelet translocation velocity was determined in vitro according to a modified method of Savage et al. (1Savage B. Saldivar E. Ruggeri Z.M Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar). Platelets were considered to have translocated when their spatial displacement was greater than one cell diameter. Platelet motion was analyzed off-line utilizing Leica confocal software. Briefly, a sequential stack of images over a 37.5-s time frame were projected using Leica acquisition software, and the xy coordinates of the centroid of individual platelets were determined every 0.576 s. Mean platelet translocation velocities were determined over the entire 37.5-s scan. Isolated human platelets were loaded with Oregon Green BAPTA and Fura Red as per established protocol (24Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Guiliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Calcium dye loaded platelets were subsequently incubated in PWB with 10 μm NP-EGTA (Molecular Probes) for 30 min at 37 °C. NP-EGTA-treated platelets were subsequently washed once with PWB and resuspended in PWB prior to experimentation. The potential effects of the NP-EGTA precursor loading (prior to photolysis) on platelet function were assessed using standard aggregometry following stimulation of the cells with 1–12.5 μm ADP or 0.1–1 units/ml thrombin. Incubation with 10 μm NP-EGTA did not modify the aggregation of platelets to these agonists relative to untreated controls. The behavior of NP-EGTA-loaded platelets was assessed under flow conditions (1,800·s−1) at the surface of immobilized human vWf (100 μg/ml) as per established protocols. NP-EGTA-loaded platelets exhibited translocation and adhesive properties equivalent to untreated controls. NP-EGTA uncaging was carried out following 18 s of reconstituted blood flow via exposure of platelets to a near UV (300–400-nm) light source generated by a 100-watt mercury lamp directed through the optical path of a Leica DMIRBE confocal microscope, for an interval of 0.6 s. Control studies were carried out with unloaded control platelets and demonstrated that the brief (0.6-s) UV exposure did not lead to photodynamic damage or activation of the platelets under flow. Recent studies examining shear-dependent platelet adhesion on immobilized vWf under flow have demonstrated a role for both the GPIb/V/IX and integrin αIIbβ3 receptor complexes in regulating cytosolic calcium flux (7Yuan Y. Ulsemer P. Cranmer S.L. Yap C.L. Nesbitt W.S. Harper I. Mistry N. Dopheide S.M. Hughan S.C. Williamson D. de la Salle C. Salem H.H. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 36241-36251Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 8Kuwahara M. Sugimoto M. Tsuji S. Miyata S. Yoshioka A. Blood. 1999; 94: 1149-1155Crossref PubMed Google Scholar). However, the temporal relationship and mechanisms by which these receptors regulate cytosolic calcium (Δ[Ca2+]c) remain incompletely understood. In this study, we have employed a confocal-based dual dye ratiometric Ca2+ assay to accurately quantitate real-time cytosolic Ca2+ flux during platelet adhesion under flow (24Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Guiliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In initial studies, Oregon Green BAPTA and Fura Red-loaded human platelets were perfused through human vWf-coated (100 μg/ml) microcapillary tubes at a shear rate of 1,800·s−1, and the relationship between cytosolic calcium flux and platelet translocation behavior was examined. Fig. 1A demonstrates that the platelet population exhibited a broad range of Δ[Ca2+]c, ranging from 0 to 1,200 nm, and translocation velocities between 0 and 20 μm·s−1, similar to those reported by Savage et al. (1Savage B. Saldivar E. Ruggeri Z.M Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar). Detailed single-cell analysis revealed that platelets exhibit three broad calcium response subclasses that differ with respect to the magnitude and dynamics of their calcium flux. The first subclass consists of platelets with a relatively low cytosolic calcium content (Δ[Ca2+]c <20 nm). These platelets were characterized by a rapid rate of translocation across the vWF surface and minimal calcium oscillations (Fig. 1B). The second subclass exhibited a moderately elevated (intermediate) Δ[Ca2+]c, ranging from 20 to 65 nm, which underwent minor oscillations; these platelets displayed a reduced rate of translocation that was stop-start in nature (Fig.1B). The third and final subclass of platelets displayed elevated oscillatory Δ[Ca2+]c, ranging from 65 up to 1200 nm. The defining quality of platelets falling into this high range calcium response subclass was the pulsatile nature of the calcium flux, with cells undergoing rapid base-line to peak oscillations. The translocation behavior of these platelets was characterized by extended periods of stationary adhesion on the matrix surface (Fig. 1B). To examine in more detail the correlation between Δ[Ca2+]c and translocation behavior, platelets were pretreated with the high affinity calcium chelator DM-BAPTA prior to perfusion through vWf-coated microcapillary tubes. DM-BAPTA treatment resulted in an approximately 10-fold increase in platelet translocation velocity, from a mean velocity of 6.2 μm·s−1 for controls up to 61.4 μm·s−1for DM-BAPTA-treated platelets (Fig. 1A). In contrast, when the cytosolic concentration of calcium was artificially elevated, by pretreating platelets with increasing concentrations of the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (Tg), there was a significant concentration-dependent decrease in mean platelet translocation velocity. Single cell analysis of thapsigargin-treated platelets revealed a close inverse correlation (R = 0.94) between the cytosolic calcium level and the translocation velocity of individual platelets, with mean platelet translocation velocities approaching 0 μm·s−1 as the mean Δ[Ca2+]c approached 100 nm (Fig.1C). In control studies, we examined the potential role for ADP, thromboxane A2, or trace amounts of thrombin in platelet calcium signaling, by pretreating platelets with apyrase (2 units/ml), aspirin (1.5 mm), and/or hirudin (200 units/ml) prior to perfusion through vWf-coated microcapillary tubes. Consistent with previous findings (24Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Guiliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), none of these inhibitors, either alone or in combination, modified the distribution of calcium events initiated by vWf engagement of GPIb and integrin αIIbβ3 at 1,800·s−1 (data not shown). Our recent studies have demonstrated that platelets forming irreversible adhesion contacts on a vWf matrix, under static or flow conditions, exhibit a sustained oscillatory calcium response (24Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Guiliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). To investigate the relative roles of GPIb/V/IX and integrin αIIbβ3 in regulating these calcium changes, platelets were pretreated with vehicle, Aggrastat, or c7E3 Fab prior to performing adhesion studies on vWf. As demonstrated in Fig.2A, platelets forming irreversible adhesion contacts with vWf under static conditions elicited sustained oscillatory calcium responses. However, blocking ligand binding to integrin αIIbβ3completely abolished these sustained oscillations, with all cells exhibiting distinct calcium spikes (Fig. 2A). Several lines of evidence demonstrate that these calcium spikes are elicited as a result of the vWf-GPIb interaction, rather than secondary to the release of ADP or thromboxane A2. First, pretreating platelets with apyrase or aspirin had no inhibitory effect on the generation of these calcium spikes (data not shown). Second, blocking ligand binding to GPIb abolished all calcium responses (data not shown). Third, pretreating platelets with ristocetin, a cationic modulator that increases the affinity of the vWf-GPIb interaction, increased the proportion of cells that elicited these isolated calcium spikes (Fig. 2C) but did not affect the amplitude of the Ca2+ transients (mean Δ[Ca2+]c = 305 ± 146 nm; n = 40) (Fig.2B). These studies suggest that even in the absence of shear, GPIb binding to vWf is sufficient to induce a transient Ca2+ signal. We have previously demonstrated that shear increases the proportion of platelets exhibiting a GPIb-dependent calcium signal (7Yuan Y. Ulsemer P. Cranmer S.L. Yap C.L. Nesbitt W.S. Harper I. Mistry N. Dopheide S.M. Hughan S.C. Williamson D. de la Salle C. Salem H.H. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 36241-36251Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). To investigate the effects of shear on the magnitude and duration of calcium signals initiated by GPIb/V/IX and integrin αIIbβ3, control or Aggrastat-treated platelets were perfused through vWf-coated microcapillary tubes at a shear rate of 1,800·s−1. Platelets forming stationary adhesion contacts under high shear exhibited a sustained oscillatory calcium response similar in magnitude and duration to that observed under static conditions (data not shown). Integrin αIIbβ3 blockade under these conditions resulted in almost complete inhibition of high range Δ[Ca2+]c (Fig.3A), with a concomitant (5-fold) increase in platelet translocation velocity, from a mean of 6.2 μm·s−1 in control platelets up to 34.1 μm·s−1 in Aggrastat-treated platelets (data not shown). To examine specifically the effects of shear on GPIb-derived calcium signals, independent of platelet translocation, Aggrastat-treated platelets were perfused through vWf-coated microcapillary tubes in the presence of ristocetin. Under these experimental conditions, the vWf-GPIb interaction sustains stationary platelet adhesion under flow (1800·s−1), independent of integrin αIIbβ3 (data not shown). In control studies, we demonstrated that the presence of ristocetin did not have any significant effect on the overall magnitude and duration of the sustained oscillatory calcium response initiated by vWf engagement of GPIb/V/IX and integrin αIIbβ3(compare Fig. 3C with Fig. 2, A andB). Analysis of Aggrastat-treated platelets at 1,800·s−1 revealed that ∼67% of adherent cells displayed discrete calcium transients or "spikes," that exhibited a mean Δ[Ca2+]c of 391 ± 182.9 nm (maximum Δ[Ca2+]c = 896 nm; n = 41) (Fig. 3, B andC). Thus, regardless of the experimental conditions, the vWf-GPIb interaction appears to elicit transient Ca2+spikes that are distinct from the complex oscillatory response initiated by integrin αIIbβ3 engagement of vWf. To investigate the potential importance of transient calcium spikes in regulating platelet adhesion dynamics under flow, we examined calcium response profiles of individual translocating platelets. Detailed analysis of platelets undergoing high range Ca2+ responses under shear conditions revealed a subset of cells that exhibited elevated but transient calcium responses (Fig.4A). As demonstrated in Fig.4A, these transient calcium spikes coincided with a brief period of stationary adhesion, which was followed by a return to surface translocation following a decline in Δ[Ca2+]c toward 100 nm. In contrast, all platelets exhibiting sustained oscillatory calcium responses maintained stationary adhesion contacts with the vWf matrix (Fig. 4A). To examine more directly the role of calcium spikes in initiating transient platelet arrest, we developed a caged-calcium platelet activation assay. NP-EGTA is a caged calcium chelator that displays a marked increase in its Kd for Ca2+ upon photolysis with near UV light (300–400 nm). This reagent can effectively be used to release a relatively large concentration of free Ca2+ in the cytosol within milliseconds of UV activation. In control studies, we demonstrated that loading platelets with 10 μm NP-EGTA alone, independent of UV exposure, had no effect on the ability of platelets to adhere to thrombogenic surfaces or to aggregate in response to soluble agonist stimulation. However, following brief exposure to UV irradiation, these cells rapidly released caged calcium (data not shown). To investigate the effects of transient calcium spikes on platelet adhesion under flow, NP-EGTA-loaded platelets were perfused through vWf-coated microcapillary tubes at a shear rate of 1,800·s−1. Exposure of these cells to UV light for a period of 0.6 s resulted in the generation of a rapid spike in Δ[Ca2+]c, approaching 700–1200 nm (Fig. 4B). Examination of the translocation properties of these platelets demonstrated that stationary adhesion was tightly controlled by the onset of the calcium spikes (Fig. 4B). Platelet adhesion under these experimental conditions was mediated through integrin αIIbβ3, since it was completely prevented by pretreating platelets with c7E3 Fab or Aggrastat (Fig.4B). In control studies, we demonstrated that the effects of uncaged calcium on stationary platelet adhesion were likely to be direct rather than secondary to release of ADP or thromboxane A2, since pretreating platelets with apyrase or aspirin had no effect on UV light-induced platelet adhesion (data not shown). To investigate further the relationship between sustained calcium oscillations and firm cell adhesion, NP-EGTA-loaded platelets were pretreated with a suboptimal concentration of Tg (1 nm) 2 min prior to perfusion through vWf-coated microcapillary tubes. Pretreatment with 1 nm Tg had minimal effect on platelet translocation behavior at a shear rate of 1,800·s−1prior to UV photolysis (Fig. 4C). Triggering of a Ca2+ spike by UV photolysis of NP-EGTA led to an equivalent transient Ca2+ elevation as that observed for control platelets not incubated with Tg (Fig. 4C). Tg pretreatment partially blocked the reuptake of the NP-EGTA-elicited Ca2+spikes and led to the onset of an oscillatory Ca2+ response (Δ[Ca2+]c >100 nm) (Fig.4C) in the entire platelet population (data not shown). The prolonged oscillatory Ca2+ response in the presence of Tg following UV exposure directly correlated with an increased duration of stationary adhesion (Fig. 4C). This stationary adhesion was mediated by integrin αIIbβ3, since it was completely blocked by pretreating platelets with c7E3 or Aggrastat (Fig. 4C). Taken together, these data suggest that transient Ca2+ spikes may serve to initiate integrin αIIbβ3 activation and transient cell arrest; however, irreversible adhesion appears to be dependent on sustained integrin αIIbβ3-dependent calcium oscillations. Our recent studies have defined an important role for transmembrane calcium influx and PI 3-kinase in potentiating sustained Ca2+ oscillations and integrin αIIbβ3 activation during platelet adhesion to immobilized vWf under flow (24Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Guiliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26Yap C.L. Anderson K.E. Dopheide S.M. Salem H.H. Jackson S.P. Blood. 2002; 99 (in press)Crossref PubMed Scopus (108) Google Scholar). To investigate the contribution of transmembrane calcium influx to GPIb and integrin αIIbβ3-dependent calcium signaling, in vitro flow studies were performed in the presence or absence of EGTA. As demonstrated in Fig.5A, EGTA markedly reduced integrin αIIbβ3-dependent calcium signals at 1,800·s−1, resulting in an 86% reduction in the frequency of Δ[Ca2+]c events occurring above 100 nm in the platelet population. In contrast, chelating extracellular calcium had no inhibitory effect on the magnitude or duration of GPIb-dependent calcium spikes (n = 40) (Fig. 5B). Significantly, even in the absence of extracellular calcium, integrin αIIbβ3 engagement of vWf was still able to induce a sustained oscillatory calcium response (data not shown), indicating that ligand binding to this receptor can initiate calcium release from internal stores. To investigate the role of PI 3-kinase for GPIb and integrin αIIbβ3-dependent calcium signaling, platelets were pretreated with concentrations of wortmannin (100 nm) or LY294002 (25 μm) that effectively abolish PI 3-kinase activation in vWf-stimulated platelets (26Yap C.L. Anderson K.E. Dopheide S.M. Salem H.H. Jackson S.P. Blood. 2002; 99 (in press)Crossref PubMed Scopus (108) Google Scholar). As demonstrated in Fig. 6A, both inhibitors significantly inhibited integrin αIIbβ3-dependent high range calcium oscillations, resulting in a 98% reduction in the frequency of Δ[Ca2+]c events occurring above 100 nm in the platelet population. In contrast, neither wortmannin nor LY294002 had any significant effect on transient calcium spikes initiated by the vWf-GPIb interaction (Fig. 6B). Consistent with our previous findings, inhibition of sustained calcium oscillations resulted in an inability of the platelets to form irreversible adhesion contacts under flow (data not shown). These findings suggest that PI 3-kinase involvement in shear-dependent platelet adhesion is primarily linked to integrin αIIbβ3, not GPIb-dependent calcium signals. The studies reported here have demonstrated the existence of two distinct, cooperative calcium signaling mechanisms utilized by GPIb/V/IX and integrin αIIbβ3 to regulate platelet-adhesive behavior under flow (Fig.7). A significant finding from these studies is that GPIb/V/IX engagement of vWf elicits a transient calcium response that may serve to initiate integrin αIIbβ3 activation; however, this calcium signal appears insufficient to maintain sustained platelet adhesion in the shear field. Our studies suggest that subsequent integrin αIIbβ3 engagement of vWf triggers outside-in signaling events linked to the initiation and propagation of sustained oscillatory calcium flux that is necessary for stable platelet adhesion. By examining real time changes in cytosolic calcium during platelet translocation, we have been able to establish a close correlation between cytosolic calcium fluctuations and the stop-start nature of platelet translocation. In particular, we have been able to identify platelets that exhibit transient calcium oscillations that are associated with intermittent phases of stationary adhesion, often lasting 10–20 s or more. In all cells, the transition from stationary adhesion to surface translocation corresponded to a drop in the cytosolic calcium, approaching 100 nm. These studies suggest that a critical calcium threshold must be reached and maintained for platelets to sustain stable adhesion contacts in a shear field. Furthermore, we have demonstrated that this calcium-dependent regulation of platelet translocation behavior is mediated through the reversible activation of integrin αIIbβ3, establishing for the first time an important role for this receptor in regulating platelet translocation dynamics under flow. Our studies have also provided new insight into the relative contribution of GPIb and integrin αIIbβ3 outside-in signaling toward vWf-induced calcium flux. The traditional model for platelet activation by vWf, based primarily on studies of shear-induced platelet aggregation using a cone-and-plate viscometer, had suggested that GPIb-induced transmembrane calcium influx represented the critical proximal signaling step for subsequent ADP release and integrin αIIbβ3 activation. However, recent studies from our laboratory have demonstrated that vWf binding to GPIb is sufficient to induce intracellular calcium mobilization, independent of transmembrane calcium influx and ADP release, as a necessary event for subsequent integrin αIIbβ3 activation (7Yuan Y. Ulsemer P. Cranmer S.L. Yap C.L. Nesbitt W.S. Harper I. Mistry N. Dopheide S.M. Hughan S.C. Williamson D. de la Salle C. Salem H.H. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 36241-36251Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar,24Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Guiliano S. Kulkarni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In this report, we demonstrate the cooperative relationship between GPIb and integrin αIIbβ3 in regulating cytosolic calcium flux and, furthermore, demonstrate that the calcium signaling mechanisms operating downstream of GPIb/V/IX and integrin αIIbβ3 are distinct, in that the former are primarily due to calcium release from internal stores, whereas the latter are dependent on both intracellular calcium mobilization and transmembrane calcium influx. The reason for this difference remains to be established but may be related to differences in the duration and magnitude of calcium mobilization. Calcium store emptying is a major stimulus for transmembrane calcium influx, and it is conceivable that integrin αIIbβ3-dependent sustained calcium flux leads to more extensive depletion of intracellular calcium stores in comparison with the transient calcium spikes initiated by GPIb binding. Alternatively, integrin αIIbβ3 engagement may directly activate an associated calcium influx pathway, as proposed by Brass (15Brass L.F. J. Biol. Chem. 1985; 260: 2231-2236Abstract Full Text PDF PubMed Google Scholar). This study demonstrates for the first time a role for PI 3-kinase in regulating integrin αIIbβ3 calcium signaling and, somewhat unexpectedly, does not support an important role for this kinase in GPIb signaling, at least under high shear conditions. There are two potential mechanisms by which PI 3-kinase may promote integrin αIIbβ3 activation and sustained calcium oscillations. First, while not essential for GPIb-dependent calcium flux, PI 3-kinase may function downstream of cytosolic calcium to initiate integrin αIIbβ3 activation. Alternatively, PI 3-kinase may participate in integrin αIIbβ3outside-in signaling events associated with the initiation and propagation of intracellular calcium oscillations, necessary for sustained integrin αIIbβ3 activation. While we have not formally excluded the former hypothesis, several lines of evidence suggest that the latter hypothesis is more likely. First, to our knowledge there are no precedents for PI 3-kinase signaling downstream of calcium. Second, there is a considerable body of evidence demonstrating PI 3-kinase involvement in integrin αIIbβ3 outside-in signaling. For example, the production of 3-phosphorylated phosphoinositides in thrombin-stimulated platelets is regulated downstream of integrin αIIbβ3 (27Sultan C. Plantavid M. Bachelot C. Grondin P. Breton M. Mauco G. Levy-Toledano S. Caen J.P. Chap H. J. Biol. Chem. 1991; 266: 23554-23557Abstract Full Text PDF PubMed Google Scholar). Furthermore, direct ligand binding to integrin αIIbβ3 is sufficient to induce PI 3-kinase activation and a selective increase in the cellular levels of phosphatidylinositol 3,4-bisphosphate (28Kovacsovics T.J. Bachelot C. Toker A. Vlahos C.J. Duckworth B. Cantley L.C. Hartwig J.H. J. Biol. Chem. 1995; 270: 11358-11366Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). This integrin αIIbβ3-dependent activation of PI 3-kinase has been proposed to sustain integrin αIIbβ3 activation as an important event for cytoskeletal reorganization, platelet spreading, and irreversible aggregation (28Kovacsovics T.J. Bachelot C. Toker A. Vlahos C.J. Duckworth B. Cantley L.C. Hartwig J.H. J. Biol. Chem. 1995; 270: 11358-11366Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 29Heraud J.M. Racaud-Sultan C. Gironcel D. Albiges-Rizo C. Giacomini T. Roques S. Martel V. Breton-Douillon M. Perret B. Chap H. J. Biol. Chem. 1998; 273: 17817-17823Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). It remains to be established what the molecular mechanism is by which PI 3-kinase promotes integrin αIIbβ3-dependent cytosolic calcium flux. Previous studies in platelets and a range of other cells have demonstrated that the PI 3-kinase lipid product, phosphatidylinositol 3,4,5-trisphosphate, plays a significant role in the regulation of both intracellular calcium mobilization and transmembrane calcium flux (30Pasquet J-M. Quek L. Stevens C. Bobe R. Huber M. Duronio V. Krystal G. Watson S.P. EMBO J. 2000; 19: 2793-2802Crossref PubMed Scopus (75) Google Scholar, 31Hsu A.L. Ching T.T. Sen G. Wang D.S. Bondads S. Authi K.S. Chen C.S. J. Biol. Chem. 2000; 275: 16242-16250Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 32Bae Y.S. Cantley L.G. Chen C.S. Kim S.R. Kwon K.S. Rhee S.G. J. Biol. Chem. 1998; 273: 4465-4469Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 33Falasca M. Logan S.K. Lehto V.P. Baccante G. Lemmon M.A. Schlessinger J. EMBO J. 1998; 17: 414-422Crossref PubMed Scopus (488) Google Scholar, 34Hippen K.L. Buhl A.M. D'Ambrosio D. Nakamura K. Persin C. Cambier J.C. Immunity. 1997; 7: 49-58Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). However, given previous observations that direct ligation of integrin αIIbβ3 leads to a selective increase in phosphatidylinositol 3,4-bisphosphate, it remains to be determined whether phosphatidylinositol 3,4,5-trisphosphate plays a significant role in integrin αIIbβ3-dependent calcium signaling. Finally, much of our current conceptual understanding of integrin αIIbβ3 signaling in platelets is based on studies of platelets in suspension, in which the addition of soluble stimuli is required to initially induce integrin αIIbβ3 activation (inside-out signaling). In this assay system, subsequent ligand binding events are required to generate outside-in signals, necessary to promote relatively "late" platelet functional responses, including irreversible platelet aggregation, clot retraction, and the shedding of procoagulant-rich microvesicles. Our studies suggest that the establishment of integrin αIIbβ3 outside-in calcium signaling plays a critical "early" role in the initial activation of platelets on vWf. These studies define a previously unrecognized role for integrin αIIbβ3 in driving a calcium-dependent positive feedback mechanism that plays an important role in regulating the affinity status of the integrin itself. We thank Dr. Steve Watson, Prof. Leslie Parise, and Dr. Yuping Yuan for helpful discussions and constructive advice during the preparation of the manuscript.